Human insulin-like growth factor I (IGF-I) is a circulating hormone structurally related to insulin. IGF-I is traditionally considered the major mediator of the actions of growth hormone on peripheral tissues. Naturally-occurring IGF-I consists of 70 amino acids and is also named somatomedin C and defined by SwissProt No. P01343. Use, activity and production are mentioned in, e.g., le Bouc, Y., et al., FEBS Lett. 196 (1986) 108-112; de Pagter-Holthuizen, P., et al., FEBS Lett. 195 (1986) 179-184; Sandberg Nordqvist, A. C., et al., Brain Res. Mol. Brain Res. 12 (1992) 275-277; Steenbergh, P. H., et al., Biochem. Biophys. Res. Commun. 175 (1991) 507-514; Tanner, J. M., et al., Acta Endocrinol. (Copenh.) 84 (1977) 681-696; Uthne, K., et al., J. Clin. Endocrinol. Metab. 39 (1974) 548-554; EP 0 123 228; EP 0 128 733; U.S. Pat. No. 5,861,373; U.S. Pat. No. 5,714,460; EP 0 597 033; WO 02/32449; WO 93/02695.
The regulation of IGF-I function is quite complex. In the circulation, only 0.2% of IGF-I exists in the free form whereas the majority is bound to IGF-binding proteins (IGFBP's), which have very high affinities to IGF's and modulate IGF-I function. The factor can be locally liberated by mechanisms releasing IGF-I such as proteolysis of IGFBPs by proteases.
IGF-I plays a paracrine role in the developing and mature brain (Werther, G. A., et al., Mol. Endocrinol. 4 (1990) 773-778). In vitro studies indicate that IGF-I is a potent non-selective trophic agent for several types of neurons in the CNS (Knusel, B., et al., J. Neurosci. 10 (1990) 558-570; Svrzic, D., and Schubert, D., Biochem. Biophys. Res. Commun. 172 (1990) 54-60), including dopaminergic neurons (Knusel, B., et al., J. Neurosci. 10 (1990) 558-570) and oligodendrocytes (McMorris, F. A., and Dubois-Dalcq, M., J. Neurosci. Res. 21 (1988) 199-209; McMorris, F. A., et al., Proc. Natl. Acad. Sci. USA 83 (1986) 822-826; Mozell, R. L., and McMorris, F. A., J. Neurosci. Res. 30 (1991) 382-390)). U.S. Pat. No. 5,093,317 mentions that the survival of cholinergic neuronal cells is enhanced by administration of IGF-I. It is further known that IGF-I stimulates peripheral nerve regeneration (Kanje, M., et al., Brain Res. 486 (1989) 396-398) and enhance ornithine decarboxylase activity U.S. Pat. No. 5,093,317). U.S. Pat. No. 5,861,373 and WO 93/02695 mention a method of treating injuries to or diseases of the central nervous system that predominantly affects glia and/or non-cholinergic neuronal cells by increasing the active concentration(s) of IGF-I and/or analogues thereof in the central nervous system of the patient. WO 02/32449 is directed to methods for reducing or preventing ischemic damage in the central nervous system of a mammal by administering to the nasal cavity of the mammal a pharmaceutical composition comprising a therapeutically effective amount of IGF-I or biologically active thereof. IGF-I is absorbed through the nasal cavity and transported into the central nervous system of the mammal in an amount effective to reduce or prevent ischemic damage associated with an ischemic event. EP 0874641 claims the use of an IGF-I or an IGF-II for the manufacture of a medicament for treating or preventing neuronal damage in the central nervous system, due to AIDS-related dementia, Alzheimer's disease (AD), Parkinson's Disease, Pick's Disease, Huntington's Disease, hepatic encephalopathy, cortical-basal ganglionic syndromes, progressive dementia, familial dementia with spastic parapavresis, progressive supranuclear palsy, multiple sclerosis, cerebral sclerosis of Schilder or acute necrotizing hemorrhagic encephalomyelitis, wherein the medicament is in a form for parenteral administration of an effective amount of said IGF outside the blood-brain barrier or blood-spinal cord barrier.
Reduction of brain and serum levels of free IGF-I has been related to the pathogenesis of sporadic and familial forms of AD. Furthermore, IGF-I protects neurons against Aβ-induced neurotoxicity (Niikura, T., et al., J. Neurosci. 21 (2001) 1902-1910; Dore, S., et al., Proc. Natl. Acad. Sci. USA 94 (1997) 4772-4777; Dore, S., et al., Ann. NY Acad. Sci. 890 (1999) 356-364). Recently, it was shown that peripherally administered IGF-I is capable of reducing brain Aβ levels in rats and mice (Carro, E., et al., Nat. Med. 8 (2002) 1390-1397). Furthermore, the study demonstrated that in a transgenic AD mouse model prolonged IGF-I treatment significantly reduced brain amyloid plaque load. These data strongly support the idea that IGF-I is able to reduce brain AB levels and plaque-associated brain dementia by clearing Aβ from the brain.
The recognition site of the IgA Protease is described as Yaa-Pro.!.Xaa-Pro. Yaa stands for Pro (or rarely for Pro in combination with Ala, Gly or Thr: Pro-Ala, Pro-Gly, or Pro-Thr. Xaa stands for Thr, Ser or Ala (Pohlner, J. et al., Bio/Technology 10 (1992) 799-804; Pohlner, J. et al., Nature 325 (1987) 458-462 and U.S. Pat. No. 5,427,927). Naturally cleavage sites have been identified by Wood, S. G. and Burton, J., Infect Immun. 59 (1991) 1818-1822. Synthetic peptide substrates for the immunoglobulin A1 protease from Neisseria gonorrhoeae (type 2) are the autoproteolytic sites Lys-Pro-Ala-Pro.!.Ser-Pro (SEQ ID NO: 22), Val-Ala-Pro-Pro.!.Ser-Pro (SEQ ID NO: 23), Pro-Arg-Pro-Pro.!.Ala-Pro (SEQ ID NO: 24), Pro-Arg-Pro-Pro.!.Ser-Pro (SEQ ID NO: 25), Pro-Arg-Pro-Pro.!.Thr-Pro (SEQ ID NO: 26) and the IgA1 Cleavage Sites Pro-Pro-Thr-Pro.!.Ser-Pro (SEQ ID NO: 27) and Ser-Thr-Pro-Pro.!.Thr-Pro (SEQ ID NO: 28).
WO 2006/066891 discloses conjugates consisting of an insulin-like growth factor-1 (IGF-I) and one or two poly(ethylene glycol) group(s), characterized in that said IGF-I has an amino acid alteration at up to three amino acid positions 27, 37, 65, 68 of the wild-type IGF-I amino acid sequence so that one or two of said amino acids is/are lysine and amino acid 27 is a polar amino acid but not lysine, is conjugated via the primary amino group(s) of said lysine(s) and said poly(ethylene glycol) group(s) have an overall molecular weight of from 20 to 100 kDa. Such conjugates are useful for the treatment of neurodegenerative disorders like Alzheimer's Disease.
WO2006/074390 refers to IGF-I variants and fusion proteins comprising IGF-I variants and certain fusion components. WO 2006/074390 refers to certain IGF-I variants.
Methods for the recombinant production of IGF-I via a fusion protein are known, e.g., from EP0155655 and U.S. Pat. No. 5,158,875. However microheterogenity of recombinantly produced IGGF-I is often found (Forsberg, G. et. al., Biochem. J. 271 (1990) 357-363).